Actively replicating gut bacteria identified by 5-ethynyl-2’-deoxyuridine (EdU) click chemistry and cell sorting

ABSTRACT

The composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2’-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labeled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro. We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium, suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

Plain Language Summary

The bacteria that live in our guts are known to influence our intestinal and overall health. Though we know a lot about which kinds of bacteria are in our guts, we still don’t know much about which bacteria are actually alive and growing. This is important to know, because bacteria that are growing, or replicating, are more likely to impact our health than bacteria which are not replicating. Our research group aimed to address this issue by developing a new technique that can identify which gut bacteria are actively replicating. We first tested this technique on specific gut bacteria, and then we made sure the technique worked when it was used on the gut bacteria of mice. By using this technique, we identified several types of mouse gut bacteria that were actively replicating. We also demonstrated one possible application of this technique by using it to identify mouse gut bacteria that were able to replicate after they were grown with antibiotics. Overall, we have introduced a new technique to identify replicating gut bacteria and show how it can be used to increase our knowledge on which bacteria are growing in the gut. This technique will help us identify which bacteria may be more important to our health due to their active growth.

KEYWORDS:

• Click chemistry
• gut microbiota
• bacterial physiology
• bacterial replication
• EdU
• 5-ethynyl-2‘-deoxyuridine
• flow cytometry
• cell sorting

Disclosure statement

The authors report there are no competing interests to declare.

Authors’ contributions

ETB designed, performed, analyzed, and interpreted experiments. CH aided with the bacterial isolate experiments. CFM helped develop the project, obtained funding, and helped design experiments and interpret the data. ETB and CFM edited the manuscript and approved the final draft.

Data availability statement

Bacterial 16S rRNA gene sequencing data can be accessed on the NCBI SRA database, accession number PRJNA855973. Code related to the analysis has been deposited in GitHub (https://github.com/evetb). Raw microscopy and flow cytometry data have been deposited in FigShare (https://doi.org/10.6084/m9.figshare.c.6103014.v1).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2180317.

Additional information

Funding

This work was supported by a Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship - Masters (CGS M, Frederick Banting and Charles Best) and Ferrings Pharmaceuticals Fellowship from McGill University to ETB. This work was also supported by a CIHR transition grant to CFM (PJT-149098), the Kenneth Rainin Foundation (Innovator Award 2016-1280) and the McGill University Health Centre Foundation (Owens Catchpaugh IBD Research Fellowship). CFM is a Tier 2 Canada Research Chair in Gut Microbial Physiology (950-230748 X-242502).